Microbial expression of a gene for human growth hormone

ABSTRACT

Described are methods and means for the construction and microbial expression of quasi-synthetic genes arising from the combination of organic synthesis and enzymatic reverse transcription from messenger RNA sequences incomplete from the standpoint of the desired protein product. Preferred products of expression lack bio-inactivating leader sequences common in eukaryotic expression products but problematic with regard to microbial cleavage to yield bioactive material. Illustrative is a preferred embodiment in which a gene coding for human growth hormone (useful in, e.g., treatment of hypopituitary dwarfism) is constructed and expressed.

This application is a division of application Ser. No. 55,126, filed7/5/79.

BACKGROUND OF THE INVENTION Genetic Expression

The DNA (deoxyribonucleic acid) of which genes are made comprises bothprotein-encoding or "structural" genes and control regions that mediatethe expression of their information through provision of sites for RNApolymerase binding, information for ribosomal binding sites, etc.Encoded protein is "expressed" from its corresponding DNA by a multistepprocess within an organism by which:

1. The enzyme RNA polymerase is activitated in the control region(hereafter the "promoter") and travels along the structural gene,transcribing its encoded information into messenger ribonucleic acid(mRNA) until transcription of translatable mRNA is ended at one or more"stop" codons.

2. The mRNA message is translated at the ribosomes into a protein forwhose amino acid sequence the gene encodes, beginning at a translation"start" signal, most commonly ATG (which is transcribed "AUG" andtranslated" f-methionine").

In accordance with the genetic code, DNA specifies each amino acid by atriplet or "codon" of three adjacent nucleotides individually chosenfrom adenosine, thymidine, cytidine and guanine or, as used herein, A,T, C, or G. These appear in the coding strand or coding sequence ofdouble-stranded ("duplex") DNA, whose remaining or "complementary"strand is formed of nucleotides ("bases") which hydrogen bond to theircomplements in the coding strand. A complements T, and C complements G.These and other subjects relating to the background of the invention arediscussed at length in Benjamin Lewin, Gene Expression 1, 2 (1974) and 3(1977), John Wiley and Sons, N.Y. This and the other publicationsalluded to herein are incorporated by reference.

DNA Cleavage and Ligation

A variety of techniques are available for DNA recombination, accordingto which adjoining ends of separate DNA fragments are tailored in oneway or another to facilitate ligation. The latter term refers to theformation of phosphodiester bonds between adjoining nucleotides, mostoften through the agency of the enzyme T4 DNA ligase. Thus, blunt endsmay be directly ligated. Alternatively, fragments containingcomplementary single strands at their adjoining ends are advantaged byhydrogen bonding which positions the respective ends for subsequentligation. Such single strands, referred to as cohesive termini, may beformed by the addition of nucleotides to blunt ends using terminaltransferase, and sometimes simply by chewing back one strand of a bluntend with an enzyme such λ-exonuclease. Again, and most commonly, resortmay be had to restriction endonucleases (hereafter, "restrictionenzymes"), which cleave phosphodiester bonds in and around uniquesequences of nucleotides of about 4-6 base pairs in length ("restrictionsites"). Many restriction enzymes and their recognition sites are known.See, e.g., R. J. Roberts, CRC Critical Reviews in Biochemistry, 123(November 1976). Many make staggered cuts that generate shortcomplementary single-stranded sequences at the ends of the duplexfragments. As complementary sequences, the protruding or "cohesive" endscan recombine by base pairing. When two different molecules are cleavedwith this enzyme, crosswise pairing of the complementary single strandsgenerates a new DNA molecule, which can be given covalent integrity byusing ligase to seal the single strand breaks that remain at the pointof annealing. Restriction enzymes which leave coterminal or "blunt" endson duplex DNA that has been cleaved permit recombination via, e.g., T4ligase with other blunt-ended sequences.

Cloning Vehicles and Recombinant DNA

For present purposes, a "cloning vehicle" is an extrachromosomal lengthof duplex DNA comprising an intact replicon such that the vehicle can bereplicated when placed within a unicellular organism ("microbe") bytransformation. An organism so transformed is called a "transformant".Presently, the cloning vehicles commonly in use are derived from virusesand bacteria and most commonly are loops of bacteria DNA called"plasmids".

Advances in biochemistry in recent years have led to the construction of"recombinant" cloning vehicles in which, for example, plasmids are madeto contain exogenous DNA. In particular instances the recombinant mayinclude "heterologous" DNA, by which is meant DNA that codes forpolypeptides ordinarily not produced by the organism susceptible totransformation by the recombinant vehicle. Thus, plasmids are cleavedwith restriction enzymes to provide linear DNA having ligatable termini.These are bound to an exogenous gene having ligatable termini to providea biologically functional moiety with an intact replicon and aphenotypical property useful in selecting transformants. The recombinantmoiety is inserted into a microorganism by transformation and thetransformant is isolated and cloned, with the object of obtaining largepopulations that include copies of the exogenous gene and, in particularcases, with the further object of expressing the protein for which thegene codes. The associated technology and its potential applications arereviewed in extenso in the Miles International Symposium Series 10:Recombinant Molecules: Impact on Science and Society, Beers and Bosseff,eds., Raven Press, N.Y. (1977).

Recombinant DNA Expression

Aside from the use of cloning vehicles to increase the supply of genesby replication, there have been attempts, some successful, to actuallyexpress proteins for which the genes code. In the first such instance agene for the brain hormone somatostation under the influence of the lacpromotor was expressed in E. Coli bacteria. K. Itakura et al, Science198, 1056 (1977). More recently, the A and B chains of human insulinwere expressed in the same fashion and combined to form the hormone. D.V. Goeddel et al., Proc. Nat'l. Acad. Sci., USA 76, 106 (1979). In eachcase the genes were constructed in their entirety by synthesis. In eachcase, proteolytic enzymes within the cell would apparently degrade thedesired product, necessitating its production in conjugated form, i.e.,in tandem with another protein which protected it bycompartmentalization and which could be extracellulary cleaved away toyield the product intended. This work is described in the followingpublished British patent specifications of the assignee of the presentapplication: GB Nos. 2007 675 A; 2 007 670 A; 2 007 676 A; and 2 008 123A.

While the synthetic gene approach has proven useful in the several casesthus far discussed, real difficulties arise in the case of far largerprotein products, e.g., growth hormone, interferon, etc., whose genesare correspondingly more complex and less susceptible to facilesynthesis. At the same time, it would be desirable to express suchproducts unaccompanied by conjugate protein, the necessity of whoseexpression requires diversion of resources within the organism bettercommitted to construction of the intended product.

Other workers have attempted to express genes derived not by organicsynthesis but rather by reverse transcription from the correspondingmessenger RNA purified from tissue. Two problems have attended thisapproach. To begin with, reverse transcriptase may stop transcriptionfrom mRNA short of completing cDNA for the entire amino acid sequencedesired. Thus, for example, Villa-Komaroff et al obtained cDNA for ratproinsulin which lacked codons for the first three amino acids of theinsulin precursor. Proc. Nat'l. Acad. Sci., USA 75 3727 (1978). Again,reverse transcription of mRNA for polypeptides that are expressed inprecursor form has yielded cDNA for the precursor form rather than thebioactive protein that results when, in a eukaryotic cell, leadersequences are enzymatically removed. Thus far, no bacterial cell hasbeen shown to share that capability, so that mRNA transcripts haveyielded expression products containing the leader sequences of theprecursor form rather than the bioactive protein itself. Villa-Komaroff,supra (rat proinsulin); P. H. Seeburg et al, Nature 276, 795 (1978) (ratpregrowth hormone).

Finally, past attempts by others to bacterially express hormones (ortheir precursors) from mRNA transcripts have on occasion led only to theproduction of conjugated proteins not apparently amenable toextra-cellular cleavage, e.g., Villa-Komaroff, supra,(penicillinase-proinsulin); Seeburg, supra (beta-lactamase-pregrowthhormone).

Human Growth Hormone

Human growth hormone ("HGH") is secreted in the human pituitary. Itconsists of 191 amino acids and, with its molecular weight of about21,500, is more than three times as large as insulin. Until the presentinvention, human growth hormone could be obtained only by laboriousextraction from a limited source--the pituitary glands of humancadavers. The consequent scarcity of the substance has limited itsapplications to the treatment of hypopituitary dwarfism, and even herereliable estimates suggest that human-derived HGH is available insufficient quantity to serve not more than about 50% of afflictedsubjects.

In summary, a need has existed for new methods of producing HGH andother polypeptide products in quantity, and that need has beenparticularly acute in the case of polypeptides too large to admit oforganic synthesis or convenient synthesis of genes from which thepeptide could be expressed. Expression of mammalian hormones from mRNAtranscripts has offered the promise of side-stepping difficulties thatattend the synthetic approach, but until the present has permitted onlymicrobial production of bio-inactive conjugates from which the desiredhormone could not practicably be cleaved.

SUMMARY OF THE INVENTION

The present invention provides methods and means for expressingquasi-synthetic genes wherein reverse transcription provides asubstantial portion, preferably a majority, of the coding sequencewithout laborious resort to entirely synthetic construction, whilesynthesis of the remainder of the coding sequence affords a completedgene capable of expressing the desired polypeptide unaccompanied bybio-inactivating leader sequences or other extraneous protein.Alternatively, the synthetic remainder may yield a proteolysis-resistantconjugate so engineered as to permit extra-cellular cleavage ofextraneous protein, yielding the bioactive form. The inventionaccordingly makes available method and means for microbial production ofnumerous materials hitherto produced only in limited quantity by costlyextraction from tissue, and still others previously incapable ofindustrial manufacture. In its most preferred embodiment the inventionrepresents the first occasion in which a medically significantpolypeptide hormone (human growth hormone) has been bacteriallyexpressed while avoiding both intracellular proteolysis and thenecessity of compartmentalizing the bioactive form in extraneous proteinpending extracellular cleavage. Microbial sources for human growthhormone made available by the invention offer, for the first time, amplesupplies of the hormone for treatment of hypopituitary dwarfism,together with other applications heretofore beyond the capacity oftissue-derived hormone sources, including diffuse gastric bleeding,pseudarthrosis, burn therapy, wound healing, dystrophy and boneknitting.

The manner in which these and other objects and advantages of theinvention may be obtained will appear more fully from the detaileddescription which follows, and from the accompanying drawings relatingto a preferred embodiment of the invention, in which:

FIG. 1 depicts the synthetic scheme for construction of a gene fragmentcoding for the first 24 amino acids of human growth hormone, togetherwith the start signal ATG and linkers used in cloning. The arrows in thecoding or upper strand ("U") and in the complementary or lower strand("L") indicate the oligonucleotides joined to form the depictedfragment;

FIG. 2 depicts joinder of the "U" and "L" oligonucleotides to form thegene fragment of FIG. 1, and its insertion in a plasmid cloning vehicle;

FIG. 3 illustrates the DNA sequence (coding strand only) of the Hae IIIrestriction enzyme fragment of a pituitary mRNA transcript, with thenumbered amino acids of human growth hormone for which they code. Keyrestriction sites are indicated, as in DNA (following "stop") foruntranslated mRNA;

FIG. 4 illustrates the construction of a cloning vehicle for a genefragment coding for the amino acids of human growth hormone notsynthetically derived, and the construction of that gene fragment ascomplementary DNA by reverse transcription from mRNA isolated from ahuman pituitary source; and

FIG. 5 illustrates the construction of a plasmid capable, in bacteria,of expressing human growth hormone, beginning with the plasmids of FIGS.2 and 4.

DETAILED DESCRIPTION OF THE INVENTION

The general approach of the invention involves the combination in asingle cloning vehicle of plural gene fragments which in combinationcode for expression of the desired product. Of these, at least one is acDNA fragment derived by reverse transcription from mRNA isolated fromtissue, as by the method of A. Ullrich et al, Science 196, 1313 (1977).The cDNA provides a substantial portion, and preferably at least amajority, of the codons for the desired product, while remainingportions of the gene are supplied synthetically. The synthetic and mRNAtranscript fragments are cloned separately to provide ample quantitiesfor use in the later combination step.

A variety of considerations influence distribution of codons for the endproduct as between synthetic and cDNA, most particularly the DNAsequence of complementary DNA determined as by the method of Maxam andGilbert, Proc. Nat'l Acad. Sci. USA 74, 560 (1977). Complementary DNAobtained by reverse transcription will invariably contain codons for atleast a carboxy terminal portion of the desired product, as well asother codons for untranslated mRNA downstream from the translation stopsignal(s) adjacent the carboxy terminus. The presence of DNA foruntranslated RNA is largely irrelevant, although unduly lengthysequences of that kind may be removed, as by restriction enzymecleavage, to conserve cellular resources employed in replicating andexpressing the DNA for the intended product. In particular cases, thecDNA will contain codons for the entire amino acid sequence desired, aswell as extraneous codons upstream from the amino terminus of theintended product. For example, many if not all polypeptide hormones areexpressed in precursor form with leader or signal sequences of proteininvolved, e.g., in transport to the cellular membrane. In expressionfrom eukaryotic cells, these sequences are enzymatically removed, suchthat the hormone enters the periplasmic space in its free, bioactiveform. However, microbial cells cannot be relied upon to perform thatfunction, and it is accordingly desirable to remove sequences coding forsuch signals or leader sequences from the mRNA transcript. In the courseof that removal process the translation start signal is also lost, andalmost invariably some codons for the intended product will be removedas well. The synthetic component of the quasi-synthetic gene product ofthe invention returns these latter codons, as well as supplying anew atranslation start signal where the vehicle into which the hybrid genewill ultimately be deployed itself lacks a properly positioned start.

Elimination of the leader sequence from pregrowth hormone cDNA isadvantaged by the availability of a restriction site within the growthhormone-encoding portion of the gene. The invention may nevertheless bepracticed without regard to the availability of such a site, or in anyevent without regard to the availability of a restriction sitesufficiently near the amino terminus of the desired polypeptide as toobviate the need for extensive synthesis of the gene component notderived from mRNA. Thus, in any cDNA coding for the desired polypeptideand a leader or other bioinactivating sequence the boundary between thelatter's codons and those of the mature polypeptide will appear from theamino acid sequence of the mature polypeptide. One may simply digestinto the gene coding of the peptide of choice, removing the unwantedleader or other sequence. Thus, for example, given cDNA such as:##STR1## where the endpoint of digestion is indicated by arrow, reactionconditions for exonuclease digestion may be chosen to remove the uppersequences "a" and "b", whereafter S1 nuclease digestion willautomatically eliminate the lower sequences "c" and "d". Alternativelyand more precisely, one may employ DNA polymerase digestion in thepresence of deoxynucleotide triphosphates ("d(A,T,C,G)TP"). Thus, in theforegoing example, DNA polymerase in the presence of dGTP will removesequence "c" (then stop at "G"), S1 nuclease will then digest "a"; DNApolymerase in the presence of dTTP will remove "d", (then stop at "T")and S1 nuclease will then excise "b", and so on. See generally A.Kornberg, DNA Synthesis, pp. 87-88, W. H. Freeman and Co., San Francisco(1974).

More preferably, one may simply construct a restriction site at aconvenient point within the portion of the cDNA coding for the desiredproduct, by an application of the mismatch repair synthesis technique ofA. Razin et al, Proc. Nat'l Acad. Sci. USA 75, 4268 (1978). By thistechniqud one or more bases may be substituted in an existing DNAsequence, using primers containing the mismatched substituent. At leastseven palindromic 4-base pair sequences are uniquely recognized by knownrestriction enzymes, i.e., AGCT (Alu I), CCGG (Hpa II), CGCG (Tha I),GATC (Sau 3A), GCGC (Hha), GGCC (Hae III), and TCGA (Taq I). Where thecDNA sequence contains a sequence differing from one such site in asingle base, as statistically is highly likely, repair synthesis willyield replicate cDNA containing the proper, substituent base and hencethe desired restriction site. Cleavage will delete DNA for the unwantedleader, after which synthesis will replace codons required forexpression of the complete polypeptide. E.g.,: ##STR2## It will beappreciated, of course, that longer restriction sites may be likewiseinserted where desired, or that successive repairs may create 4-basepair restriction sites where only two bases common to the site appear atthe desired point, etc.

Applications will appear in which it is desirable to express not onlythe amino acid sequence of the intended product, but also a measure ofextraneous but specifically engineered protein. Four such applicationsmay be mentioned by way of example. First, the quasi-synthetic gene mayrepresent a hapten or other immunological determinant upon whichimmunogenicity is conferred by conjugation to additional protein, suchthat vaccines are produced. See generally, G.B. patent specification 2008 123A. Again, it may be desirable for biosafety reasons to expressthe intended product as a conjugate with other, bio-inactivating proteinso designed as to permit extracellular cleavage to yield the activeform. Third, applications will be presented in which transport signalpolypeptides will precede the desired product, to permit production ofthe same by excretion through the cell membrane, so long as the signalpeptide can then be cleaved. Finally, extraneous conjugate designed topermit specific cleavage extracellularly may be employed tocompartmentalize intended products otherwise susceptible to degradationby proteases endogenous to the microbial host. At least in the latterthree applications, the synthetic adaptor molecular employed to completethe coding sequence of the mRNA transcript can additionally incorporatecodons for amino acid sequences specifically cleavable, as by enzymaticaction. For example, trypsin or will cleave specifically at arg-arg orlys-lys, etc. See GB No. 2 008 123A, supra.

From the foregoing, it will be seen that in its broadest aspect theinvention admits of manifold applications, each having in common theseattributes:

a mRNA transcript is employed which codes for a substantial portion ofthe intended polypeptide's amino acid sequence but which, if expressedalone, would produce a different polypeptide either smaller or largerthan the intended product;

protein-encoding codons for amino acid sequences other than thosecontained in the intended product, if any, are removed;

organic synthesis yields fragment(s) coding for the remainder of thedesired sequence; and

the mRNA transcript and synthetic fragment(s) are combined and disposedin a promotercontaining cloning vehicle for replication and expressionof either the intended product absent extraneous conjugated protein, orintended product conjugated to but specifically cleavable fromextraneous protein.

Of course, the expression product will in every case commence with theamino acid coded for by the translation start signal (in the case ofATG, f-methionine). One can expect this to be removed intracellularly,or in any event to leave the bioactivity of the ultimate productessentially unaffected.

Although it provides a method of general applicability in the productionof useful proteins, including antibodies, enzymes and the like, theinvention is particularly suited to the expression of mammalianpolypeptide hormones and other substances having medical applications,e.g., glucagon, gastrointestinal inhibitory polypeptide, pancreaticpolypeptide, adrenocorticotropin, beta-endorphins, interferon,urokinase, blood clotting factors, human albumin, and so on. A preferredembodiment illustrative of the invention is next discussed, in which aquasi-synthetic gene coding for human growth hormone is constructed,cloned and microbially expressed.

CONSTRUCTION AND EXPRESSION OF A CLONING VEHICLE FOR HUMAN GROWTHHORMONE 1. Cloning the Hae III fragment of the mRNA transcript (FIGS. 3and 4)

Polyadenylated mRNA for human growth hormone (HGH) was prepared frompituitary growth hormone-producing tissue by the procedure of A. Ullrichet al. Science 196, 1313 (1977) 1.5 μg of double strand ("ds") cDNA wasprepared from 5 μg of this RNA essentially as described by Wickens etal. J. Biol Chem. 253 2483 (1978), except that RNA polymerase "Klenowfragment", H. Klenow, Proc. Nat'l. Aci. USA. 65, 168 (1970), wassubstituted for DNA Polymerase I in the second strant synthesis. Therestriction pattern of HGH is such that Hae III restriction sites arepresent in the 3' noncoding region and in the sequence coding for aminoacids 23 and 24 of HGH, as shown in FIG. 3. Treatment of ds HGH cDNAwith Hae III gives a DNA fragment of 551 base pairs ("bp") coding foramino acids 24-191 of HGH. Thus, 90 ng of the cDNA was treated with HaeIII, electrophoresed on an 8% polyacryclamide gel, and the region at 550bp eluted. Approximately 1 ng of cDNA was obtained.

pBR322 prepared as in F. Bolivar et al., Gene 2 (1977) 95-113 was chosenas the cloning vehicle for the cDNA. pBR322 has been fullycharacterized, J. G. Sutcliffe, Cold Spring Harbor Symposium 43, 70(1978), is a multicopy replicating plasmid which exhibits bothampicillin and tetracycline resistance owing to its inclusion of thecorresponding genes ("Ap^(R) " and "Tc^(R) ", respectively, in FIG. 4),and which contains recognition sites for the restriction enzymes Pst I,EcoRI and Hind III as shown in the Figure.

The GC tailing method of Chang, A. C. Y. et al. Nature 275 617 (1978)was employed to combine the products of Pst I cleavage of pBR322 and ofHae III digestion of the mRNA transcript, inserting the cDNA fragmentinto the Pst I site of pBR322 in such manner as to restore the Hae IIIrestriction sites (GG↓CC) on the cDNA while restoring the Pst Irestriction sites (CTGCA↓G) at each end of the insert.

Thus, terminal deoxynucleotidyl transferase (TdT) was used to addapproximately 20 dC residues per 3' terminus as described previously,Chang, A. Y. C., supra. 60 ng of Pst I-treated pBR322 was tailedsimilarly with about 10 dG residues per 3' terminus. Annealing of thedC-tailed ds cDNA with the dG-tailed vector DNA was performed in 130 μlof 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.25 mM EDTA. The mixture washeated to 70° C., allowed to cool slowly to 37° C. (12 hours), then to20° C. (6 hours) before being used to transform E. Coli, x1776. DNAsequence analysis of the plasmid pHGH31 cloned in x1776 by the method ofMaxam and Gilbert, Proc. Nat'l. Acad. Sci. USA 74, 560 (1977) resultedin confirmation of the codons for amino acids 24-191 of HGH, as shown inFIG. 3.

E. Coli K-12 strain x1776 has the genotype F⁻ tonA53 dapD8 minA1 supE42Δ40[gal-uvrB] λ⁻ minB2 rfb-2 nalA25 oms-2 thyA57* metC65 oms-1Δ29[bioH-asd] cycB2 cycA1 hsdR2. x1776 has been certified by theNational Institutes by Health as an EK2 host vector system.

x1776 has an obligate requirement for diaminopimelic acid (DAP) andcannot synthesize the mucopolysaccharide colanic acid. It thus undergoesDAP-less death in all environments where DAP is limiting but sufficientnutrients exist to support cellular metabolism and growth. It requiresthymine or thymidine and undergoes thymineless death with degradation ofDNA when thymine and thymidine are absent from the environment but whensufficient nutrients are present to sustain metabolic activity. x1776 isextremely sensitive to bile and thus is unable to survive and thus isunable to survive passage through the intestinal tract of rats. x1776 isextremely sensitive to detergents, antibiotics, drugs and chemicals.x1776 is unable to carry out either dark or photo repair of UV-induceddamage and is thus several orders of magnitude more sensitive tosunlight than wild-type strains of E. Coli. x1776 is resistant to manytransducing phages and is conjugation deficient for inheritance of manydifferent types of conjugative plasmids due to the presence of variousmutations. x1776 is resistant to nalidixic acid, cycloserine andtrimethoprim. These drugs can therefore be added to media to permitmonitoring of the strain and to preclude transformation of contaminantsduring transformation.

x1776 grows with a generation time of about 50 min. in either L broth orPenassay broth when supplemented with 100 μg DAP/ml and 4 μgthymidine/ml and reaches final densities of 8-10×10⁸ cells/ml atstationary phase. Gentle agitation by swirling and shaking back andforth for a period of 1-2 min. adequately suspends cells withmaintenance of 100% viability. Additional details concerning x1776appear in R. Curtis et al., Molecular Cloning of Recombinant DNA,99-177, Scott and Werner, eds., Academic Press (N.Y.1977).

2. Construction and Cloning of the Synthetic Gene Fragment (FIGS. 1 and2)

The strategy for construction of the HGH quasisynthetic gene includedconstruction of a synthetic fragment comprising a blunt-end restrictioncleavage site adjacent the point at which the fragment would be joinedto the mRNA transcript. Thus, as shown in FIG. 1, the synthetic gene forthe first 24 amino acids of HGH contained a Hae III cleavage sitefollowing amino acid 23. The distal end of the synthetic fragment wasprovided with a "linker" that permitted annealing to a single strandterminal resulting from restriction cleavage in the plasmid in which themRNA transcript and synthetic fragment would ultmately be joined.

As shown in FIG. 1, and 5' ends of the duplex fragment have singlestranded cohesive termini for the Eco RI and Hind III restrictionendonucleases to facilitate plasmid construction. The methionine codonat the left end provides a site for initiation of translation. Twelvedifferent oligonucleotides, varying in size from undecamer tohexadecamer, were synthesized by the improved phosphotriester method ofCrea, R. Proc. Nat'l. Acad. Sci. USA 75, 5765 (1978). Theseoligonucleotides, U₁ to U₆ and L₁ to L₆, are indicated by arrows.

10 μg amounts of U₂ through U₆ and L₂ through L₆ were phosphorylatedusing T₄ polynucleotide kinase and (.sub.γ³² -P)ATP by a publishedprocedure. Goeddel, D. V. et al. Proc. Nat'l. Acad. Sci. USA 76, 106(1979).

Three separate T₄ ligase catalyzed reactions were performed: 10 μg of5'-OH fragment U₁ was combined with the phosphorylated U₂, L₅ and L₆ ;phosphorylated U₃, U₄, L₃ and L₄ were combined; and 10 μg of 5'-OHfragment L₁ was combined with the phosphorylated L₂, U₅ and U₆. Theseligations were carried out at 4° C. for 6 hours in 300 μl of 20 mMTris-HCl (pH 7.5), 10 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM ATP using20 units of T₄ ligase. The three ligation mixtures were then combined,20 units T₄ ligase added, and the reaction allowed to proceed for 12hours at 20° C. The mixture was ethanol precipitated and electrophoresedon a 10% polyacrylamide gel. The band migrating at 84 base pairs wassliced from the gel and eluted. pBR322 (1 μg) was treated with Eco RIand Hind III, the large fragment isolated by gel electrophoresis andligated to the synthetic DNA. This mixture was used to transform E.Coli. K-12 strain 294 (end A, thi⁻, hsr⁻, hsm_(k) ⁺). Strain 294 wasdeposited Oct. 30, 1978 in the American Type Culture Collection (ATCCNo. 31446), without restriction. Sequence analysis by the Maxam andGilbert technique, supra, on the Eco RI--Hind III insert from a plasmidpHGH3 of one transformant confirmed that depicted in FIG. 1.

3. Construction of Plasmid for the Bacterial Expression of HGH (FIG. 5)

With the synthetic fragment in pHGH3 and the mRNA transcript in pHGH31,a replicable plasmid containing both fragments was constructed using theexpression plasmid pGH6, as shown in FIG. 5. The expression plasmid,which contains tandem lac promoters, was first constructed as follows. A285 base pair Eco RI fragment containing two 95 base pair UV5 lacpromoter fragments separated by a 95 base pair heterlogous DNA fragentwas isolated from plasmid pKB268, K. Backman, et al., Cell, Vol. 13,65-71 (1978). The 285 bp fragment was inserted into the Eco RI site ofpBR322 and a clone pGH1 isolated with the promoters oriented toward andin proper reading phase with the gene for tetracycline resistance. TheEco RI site distal to the latter gene was destroyed by partial Eco RIdigestion, repair of the resulting single stranded Eco RI ends with DNApolymerase I and recircularization of the plasmid by blunt-end ligation.The resulting plasmid, pGH6, contains a single Eco RI site properlypositioned with respect to the promoter system into which the completedgene for HGH could be inserted.

To ready the synthetic fragment for combination with the RNA transcript,10 μg of pHGH3 was cleaved with Eco RI and Hae III restrictionendonucleases and the 77 base pair fragment containing coding sequencesfor HGH amino acids 1-23 was isolated from an 8% polyacrylamide gel.

The plasmid pHGH 31 (5 μg) was next cleaved with Hae III. The 551 bp HGHsequence and a comigrating 540 bp Hae III fragment of pBR322 werepurified by gel electrophoresis. Subsequent treatment with Xma I cleavedonly the HGH sequence, removing 39 base pairs from the 3' noncodingregion. The resulting 512 bp fragment was separated from the 540 bp.pBR322 Hae III piece by electrophoresis on a 6% polyacrylamide gel. 0.3μg of the 77 bp Eco RI--Hae III fragment was polymerized with T₄ ligasein a 16 μl reaction vessel for 14 hours at 4° C. The mixture was heatedto 70° C. for 5' to inactivate the ligase, then treated with Eco RI (tocleave fragments which had dimerized through their Eco RI sites) andwith Sma I (to cleave Xma I dimers), yielding a 591 bp fragment with anEco RI "cohesive" end and a Sma I "blunt" end. After purification on a6% polyacrylamide gel, approximately 30 ng of this fragment wereobtained. It should be noted that the expression plasmid pGH6 containsno Xma I recognition site. However, Sma I recognizes the same site asXma I, but cuts through the middle of it, yielding blunt ends. TheSma-cleaved terminus of the fragment derived from gHGH 31 canaccordingly be blunt end ligated into pGH6.

The expression plasmid pGH6, containing tandem lac UV5 promoters, wastreated successively with Hind III, nuclease S1, and Eco RI and purifiedby gel electrophoresis. 50 ng of the resulting vector, which had no EcoRI cohesive end and one blunt end was ligated to 10 ng of the 591 bp HGHDNA. The ligation mixture was used to transform E. Coli. X1776. Colonieswere selected for growth on tetracycline (12.5 μg/ml). It is noteworthythat insertion of the hybrid HGH gene into pGH6 destroys the promoterfor the tetracycline resistance gene, but that the tandem lac promoterpermits read-through of the structural gene for tet resistance,retaining this selection characteristic. Approximately 400 transformantswere obtained. Filter hybridization by the Grunstein--Hogness procedure,Proc. Nat'l. Acad. Sci. USA, 72, 3961 (1975) identified 12 coloniescontaining HGH sequences. The plasmids isolated from three of thesecolonies gave the expected restriction patterns when cleaved with HaeIII, Pvu II, and Pst I. The DNA sequence of one clone, pHGH107, wasdetermined.

Human growth hormone expressed by the transformants was easily detectedby direct radioimmunoassay performed on serial dilutions of lysed cellsupernatants using the Phadebas HGH PRIST kit (Pharmacia).

To demonstrate that HGH expression is under the control of the lacpromoter, pHGH107 was transformed into E. Coli strain D1210 a lac+(i^(Q)0+z+y+), a lac repressor overproducer. Meaningful levels of HGHexpression could not be detected until addition of the inducer IPTG(isopropylthiogalactoside).

Removal of the Eco RI site in pHGH107 would leave the ATG start signalthe same distance from the ribosome binding site codons of the lacpromoter as occurs in nature between those codons and the start signalfor B-galactosidase. To determine whether expression would be increasedby mimicking this natural spacing we converted pHGH107 to pHGH107-1 byopening the former with Eco RI, digesting the resulting single strandends with S1 endonuclease, and recircularizing by bluntend ligation withT4 ligase. Although the resulting plasmid proved likewise capable ofexpressing HGH, it surprisingly did so to a lesser extent than didpGH107, as shown by direct radioimmunoassay.

It will be apparent to those skilled in the art that the presentinvention is not limited to the preferred embodiment just discussed, butrather only to the lawful scope of the appended claims. Variations otherthan those hitherto discussed will be apparent, whether in the choice ofpromoter system, parental plasmid, intended polypeptide product orelsewhere. For example, other promoter systems applicable to the presentinvention include the lambda promoter, the arabinose operon (phi 80 dara) or the colicin E1, galactose, alkaline phosphatase or tryptophanpromoter systems. Host organisms for bacterial expression may be chosen,e.g., from among the Enterobacteriaceae, such as strains of Escherichiacoli and Salmonella; Bacillaceae, such as bacillus subtilis;Pneumococcus; Streptococcus; and Haemophilus influenzae. Of course, thechoice of organism will control the levels of physical containment incloning and expression that should be practiced to comply with NationalInstitutes of Health Guidelines for Recombinant DNA, 43 Fed. Reg. 60,080(1978).

While preferred for bench-scale practice of the present invention, E.Coli. x1776 could prove of limited practicality in large-scaleindustrial manufacture owing to the debilitations purposefullyincorporated in it for biosafety reasons. With appropriate levels ofphysical, rather than biological, containment such organisms as E. Coli.K-12 strain 294, supra, and E. Coli. strain RR1, genotype: Pro⁻ Leu⁻Thi⁻ R_(B) -recA+Str^(r) Lac y⁺ could be employed in larger scaleoperation. E. Coli. RR1 is derived from E. Coli HB101 (H. W. Boyer, etal, J. Mol. Bio. (1969) 41 459-472) by mating with E. Coli. K12 strainKL16 as the Hfr donor. See J. H. Miller, Experiments in MolecularGenetics (Cold Spring Harbor, New York, 1972). A culture of E. Coli. RR1was deposited Oct. 30, 1978 with the American Type Culture Collection,without restriction as to access (ATCC No. 31343). A culture of x1776was similarly deposited July 3, 1979 in the American Type CultureCollection (ATCC No. 31537). Deposits of the following were made in theAmerican Type Culture Collection July 3, 1979: plasmid pHGH107 (ATCC No.40011); plasmid pGH6 (ATCC No. 40012); strain x1776 transformed withpHGH107 (ATCC No. 31538) and E. Coli K12 strain 294 transformed withpGH6 (ATCC No. 31539).

Organisms produced according to the invention may be employed inindustrial scale fermentative production of human growth hormone,yielding product in quantities and for applications hithertounattainable. For example, transformant E. Coli cultures may be grown upin aqueous media in a steel or other fermentation vessel conventionallyaerated and agitated, in aqueous media at, e.g., about 37° C. and nearneutral pH (e.g., pH 7±0.3) supplied with appropriate nutriments such ascarbohydrate or glycerol, nitrogen sources such as ammonium sulfate,potassium sources such as potassium phosphate, trace elements, magnesiumsulfate and the like. Transformant organisms preferably exhibit one ormore selection characteristics, such as antibiotic resistance, so thatselection pressures may be imposed to discourage competitive growth ofwild-type E. coli. As an example, in the case of an ampicillin ortetracycline--reistant organism the antibiotic may be added to thefermentation medium to select out wild-type organisms which lack theresistance characteristic.

Upon completion of fermentation the bacterial suspension is centrifugedor the cellular solids otherwise collected from the broth and then lysedby physical or chemical means. Cellular debris is removed fromsupernatant and soluble growth hormone isolated and purified.

Human growth hormone may be purified from bacterial extracts using oneor a combination of (1) polyethyleneimine fractionation; (2) gelfilbration chromatography on Sephacryl S-200; (3) ion exchangechromatography on Biorex-70 resin or CM Sephadex; (4) ammonium sulphateand/or pH fractionation; and (5) affinity chromatography using antibodyresins prepared from anti-HGH IgG isolated from immunosensitized animalsor hybridomas; and desorbed under acid or slightly denaturingconditions.

We claim:
 1. A method of producing human growth hormone whichcomprises:(a) disposing a culture of bacterial transformants comprisingplasmids which, in a transformant bacterium, will express a gene forhuman growth hormone unaccompanied by the leader sequence of humangrowth hormone or other extraneous protein bound thereto within afermenter vessel comprising aeration and agitation means in an aqueous,nutriment-containing fermentation broth; (b) growing up the cultureunder aeration and agitation while supplying additional nutriments asrequired to maintain vigorous growth; (c) separating the resultingcellular mass from the fermentation broth; (d) lysing the cells to freethe contents thereof; (e) separating cellular debris from supernatant;and (f) isolating and purifying human growth hormone contained in thesupernatant.
 2. A method for producing human growth hormone which methodcomprises culturing bacterial transformants containing recombinantplasmids which will, in a transformant bacterium, express a gene forhuman growth hormone unaccompanied by the leader sequence of humangrowth hormone or other extraneous protein bound thereto, and isolatingand purifying said expressed human growth hormone.
 3. The method ofclaim 1 wherein the bacteria are transformant E. coli having a selectioncharacteristic and wherein selection pressure is applied to discouragecompetition by wild-type E. coli.